1. Field of the Invention
The present invention relates to a method for DNA or RNA quantitative analysis.
2. Background Art
The study of gene expression levels is important for drug development and the study of disease causation. However, in the biological field centered on DNA, target DNAs are often found in small copy numbers in general and are therefore amplified for measurement. However, it is increasingly demanded to accurately determine the copy numbers.
Examples of typical methods for DNA quantitation used currently include three methods, 1) electrophoresis, 2) DNA microarray (JP Patent Publication (Kokai) No. 2004-33160A (2004)), and 3) real-time PCR (Higuchi R, et al, Bio. Technology, 10: 413-417, (1992)). Of them, the electrophoresis is a quantitative method which involves electrophoresing negatively charged DNA, staining bands of separated standard and target samples, and examining the intensity ratio between them. This method requires low analysis cost but disadvantageously has low quantitative accuracy.
The DNA microarray is a quantitative method which involves competitively hybridizing target and standard samples labeled with different phosphors to a DNA chip on which oligonucleotides or cDNAs having complementary sequences thereto are immobilized, and examining a fluorescence intensity ratio. This method is capable of quantitating genes as many as several tens of thousands at a time. However, the obtained fluorescence intensity ratio is susceptible to the influences of the secondary structures of gene sequences and cross hybridization. Thus, this method disadvantageously has poor quantitative accuracy and requires huge analysis (apparatus/DNA chip/reagent) cost.
On the other hand, the real-time PCR is a quantitative method which involves simultaneously amplifying target and standard samples by PCR, while observing fluorescence emitted from the samples, and determining the original amount of the target sample from the number of PCR cycle at which the PCR product reaches a predetermined amount. To examine the amount of the PCR product, an intercalator method using SYBR GREEN or a so-called TaqMan probe method using a gene-specific probe is used. This TaqMan probe is a DNA probe comprising a quencher and a fluorophore linked via the DNA probe. The original TaqMan probe itself does not emit fluorescence. However, the probe is partially degraded during PCR amplification, and the fluorophore released therefrom emits fluorescence. This method has relatively high quantitative accuracy and a detection limit as low as approximately a few molecules. However, this method presents the problems that: it requires an expensive apparatus for monitoring PCR amplification processes in real time; dilution series (at least five different concentrations) of a standard sample having the same sequences must be prepared prior to analysis and analyzed in real time simultaneously with a target sample every time; and it requires complicated procedures and much time and labor.